Over 100 different mutations in genes for fibrillar collagens have been shown to cause genetic diseases. Byers, P. H., Trends Genet. 1990, 6, 293-300; Sykes, B., Nature 1990, 348, 18-20; Prockop, D. J., J. Biol. Chem. 1990, 265, 15349-15352; Kuivaniemi, H. et al., FASEB J. 1991, 5, 2052-2060. Moreover, the sequence of certain human collagen genes are known. Myers et al., Proc. Natl. Acad. Sci. USA 1988, 78, 3516, reported structure of a cDNA for the pro.alpha.2 of human type I procollagen and, subsequently, the structures of a series of collagen genes have been defined (see Vuorio, E. and de Crombrugghe, B., An. Rev. Biochem. 1990, 58, 837-875; Chu, M.- L. and Prockop, D. J., Extracellular Matrix and Inherited Disorders of Connective Tissue, Royce and Steinmann, Eds., Alan R. Liss, New York, 1992). Mutations in either of the two genes for type I procollagen (COL1A1 and COL1A2) cause osteogenesis imperfecta and a subset of osteoporosis; mutations in the gene for type II procollagen (COL2A1) cause chondrodysplasias and some forms of osteoarthritis; and mutations in the gene for type III procollagen (COL3A1) cause Ehlers-Danlos syndrome type IV and aneurysms. Most of the mutations of procollagen genes produce disease phenotypes by directing synthesis of structurally abnormal but partially functional pro.alpha. chains of type I, type II or type III procollagen. The partially functional pro.alpha. chains associate with and become disulfide-linked to normal proa chains. As a result, the mutant chains can have one of several major effects. Kuivaniemi, H. et al., FASEB J. 1991, 5, 2052-2060. One effect is to prevent folding of the three chains into a collagen triple helix and thereby cause degradation of both the abnormal and normal pro.alpha. chains through a process referred to as procollagen suicide. The second effect is to produce minor changes in the conformation of the collagen triple helix and thereby generate mutated monomers that interfere with self-assembly of normal monomers synthesized by the same cells.
A third effect of mutations in collagen genes is to decrease the amounts of collagen synthesized by fibroblasts and related cells. Mutations that decrease collagen synthesis, however, cause the relatively mild disease known as type I osteogenesis imperfecta. Deleterious effects of mutant collagen gene expression have been demonstrated in transgenic mice expressing mutated genes for type I procollagen. These transgenic mice developed phenotypes resembling human osteogenesis imperfecta, Stacey, A. et al., Nature 1988, 332, 131-136; Khillan, J. S. et al., J. Biol. Chem. 1991, 266, 23373-23379. Further, it has been demonstrated that transgenic mice expressing mutated genes of type II procollagen developed phenotypes resembling human chondrodysplasia. Vandenberg et al., Proc. Natl. Acad. Sci. 1991, 88, 7640-7644.
Since many heritable diseases of collagen are caused by the protein products from the mutated genes, it is believed that selective inhibition of expression of the mutated genes will be useful as a therapy for such diseases. Clinical observations have demonstrated that many patients with severe diseases caused by mutations in a collagen gene would benefit from selective inactivation of the mutant allele which directs the synthesis of mutant pro.alpha. chains. The diseases in which selective inhibition may be useful include osteogenesis imperfecta, chondrodysplasia, certain forms of osteoporosis, certain forms of aneurysms, and certain forms of osteoarthritis.
In addition, it has been recognized for many decades that many pathological conditions are caused by overproduction of collagen fibers in the forms of scars and excess fibrous tissues. For example, liver cirrhosis is a two-step process in which normal liver tissue is first destroyed by a virus or by alcohol and other toxins, and then excessive amounts of collagen fibers replace the damaged cells before normal liver cell regeneration. Idiopathic pulmonary fibrosis is a lethal condition in which, for largely unknown reasons, normal lung tissue is gradually replaced by excessive amounts of collagen fibers. Progressive systemic sclerosis (scleroderma) is a frequently lethal disease where, again for unknown reasons, skin and many internal organs become leather-like because of excessive depositions of collagen fibers. In many individuals, wounds or surgical incisions in the skin are followed by excessive depositions of collagen in the form of hypertrophic scars and keloids that present cosmetic problems and sometimes more serious consequences. Also, excessive scarring frequently occurs in normal individuals following trauma and surgical procedures. In these and related conditions, a means of specifically inhibiting collagen synthesis and deposition would be of tremendous benefit. In addition, the same means of specifically inhibiting collagen synthesis and deposition would be useful in animal husbandry. For example, most horses develop large deposits of collagen fibers resembling human keloids and called "proud flesh" following injury to the legs that can limit the effective life of both draft horses and racing thoroughbreds.
It has been demonstrated that modified antisense oligonucleotides that are complementary to specific RNAs can inhibit the expression of a number of cellular and viral genes as proteins. See Erickson, R. P., and Izant, J. G. Gene Regulation: Biology Of Antisense RNA And DNA, Vol. 1, Raven Press, New York, 1992. For example, selective inhibition of a p21 gene that differed from a normal gene by a single nucleotide has been reported. Chang, E. H. et al., Biochemistry 1991, 30, 8283-8286. Moreover, mRNA splice junctions were suitable targets for antisense nucleic acids. Kole, R. et al., Adv. Drug Delivery Rev. 1991, 6, 271-286; Munroe, S. H. EMBO J. 1988, 7, 2523-2532. Many hypotheses have been proposed to explain the mechanisms by which antisense oligonucleotides inhibit gene expression, however, the specific mechanism involved may depend on the cell type studied, the RNA targeted, the specific site on the RNA targeted, and the chemical nature of the oligonucleotide. Chiang, M.-Y. et al., J. Biol. Chem. 1991, 266, 18162-18171; Stein, C. A., and Cohen, S., Cancer Res. 1988, 48, 2659-2668.
While there is a long felt need for therapies of disorders of collagen, such need has gone unmet. Methods to selectively decrease expression of either a normal allele or a mutant allele of collagen using antisense oligonucleotides would be of tremendous benefit to those suffering from diseases of collagen.